Objective To develop a protocol of isolation and culture of human keratinocytes in order to serve as seeding cells in tissue engneering of skin and the treatment of severe burns and other traumatic skin defects or lesions. MethodsForeskin obtained from 3 to 9 year-old boys with circumcision were treated with dispase II. The epidermis was treated with trypsin to make single epidermal cells suspension. Keratinocytess were cultured in medium DMEM supplemented with serum or medium K-SFM. The rate of colony formation was calculated and the growth conditions were evaluated. ResultsThe cells grew well in both DMEM and K-SFM culture media. However, the time needed for colony formation in K-SFM medium was significantly shorter and the efficiency of colony formation was significantly higer than that in DMEM medium. ConclusionsIt is an simple and efficient method to isolate keratinocytes by dispase II and trypsin and to culture them in K-SFM medium.